The complete genome sequence of Neckar River virus confirms it to be a distinct member of the genus Tombusvirus in the family Tombusviridae

Neckar River virus (NRV), first isolated from a water sample of the Neckar River (Germany) in the 1980s, was serologically characterized as a novel tombusvirus. In this study, the complete genome sequence was determined, and an infectious full-length cDNA clone was constructed. The genome organization of NRV (DSMZ PV-0270) resembles that of tombusviruses. The genome consists of 4739 nucleotides and contains five open reading frames (ORFs) and one additional putative ORF (pX) in the 3’-terminal region. Phylogenetic analysis and sequence comparisons confirmed NRV to be a member of the species Tombusvirus neckarfluminis in the genus Tombusvirus. The infectious full-length cDNA clone was constructed using Gibson assembly and subsequent infection of Nicotiana benthamiana plants by Rhizobium radiobacter inoculation. The virus derived from the full-length cDNA clone caused symptoms resembling those caused by the wild-type virus, but slightly milder. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-023-05918-z.


Complete genome sequencing
Verification of NRV infection was done by RT-PCR.For the latter total nucleic acids were extracted using a silica-particle based method of Menzel et al. [1].Reverse transcription was carried out in a final volume of 20 µl.In a first step 3 µl of total nucleic acid extract and 1 µl primer CAATAGATTCCCACTCTGCCGAC (10 mM, salt free, Eurofins Genomics) were incubated at 99 °C for three minutes followed by a rapid cooling on ice.After that, 4 µl 5X RT-Buffer (Thermo Fisher Scientific), 1 µl RevertAid Reverse Transcriptase (20 U μl -1 , Thermo Fisher Scientific), 0.5 μl dNTPs (10 mM each, Thermo Fisher Scientific) and 10.5 µl H2O were added and incubated at 42 °C for 45 min. 1 µl of the cDNA together with 7.5 µl 2X Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific), 1 µl of primers CAATAGATTCCCACTCTGCCGAC and GCTCTCGCACTCTCAAAGAAACAG (10 mM, salt free, Eurofins Genomics) each and 4.5 µl H2O were used for the PCR reaction.The PCR program started with an initial denaturation at 98 °C for 15 s, followed by 30 cycles 98 °C for 5 s, 55 °C for 5 s and 72 °C for 15 s, finalized with 72 °C for 2 min.The amplicon covers 509 nts in the CP gene (nts 2666 -3174).
The complete genomic sequence was achieved by using the NCBI entries DQ663765 (p33/p92) and NC_038927 (CP) as templates for primer design.Subsequently, the fragment containing the gap between these two partial sequences was amplified, ligated into pTCT3 and sequenced.In a second step, a fragment located near the extreme 5'-end was amplified, ligated into pJET 1.2 blunt cloning vector via Gibson Assembly [2] and sequenced.The sense primer was derived from a tomato bushy stunt (TBSV, species Tombusvirus lycopersici) sequence.For the amplification of the 3' located fragment containing p22/p19, pX and parts of the 3' UTR the sense primer was derived from NC_038927 and the antisense primer from a TBSV sequence.Cloning and sequencing was performed as described before.By using rapid amplification of cDNA ends (RACE) [3], the sequences of the extreme 5'-and 3'-end were determined by direct sequencing of the PCR products.Primer sequences are shown in table S3.

Construction of an infectious full-length cDNA clone
To construct an infectious full-length cDNA clone, the genome of NRV was amplified as two fragments whereof fragment 1 contains 2315 nucleotides of the virus plus vector sequences of pDIVA (KX665539.1)as well as overlaps to fragment 2. Fragment 2 consists of 2585 nucleotides containing overlaps to fragment 1 and pDIVA.In the first step fragment 1 was cloned into the vector by Gibson Assembly, after which in the second step this construct was opened via PCR and fragment 2 was inserted as mentioned before.Primer sequences are shown in table S3.One putative full-length cDNA clone was completely sequenced and showed six nucleotide substitutions leading to five amino acid changes (Table S2).Transformation of R. radiobacter GV2260 was done via electroporation and bacteria were infiltrated into N. benthamiana as described in [4].S3: Oligonucleotides for the complete sequencing of NRV and construction of the infectious full-length cDNA clone.

Use Sequence
Verification   .The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 5.50 % sites).The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.This analysis involved 21 amino acid sequences.All positions containing gaps and missing data were eliminated (complete deletion option).There were a total of 300 positions in the final dataset.Evolutionary analyses were conducted in MEGA X [6].
are vector sequences, bold parts are restriction sites.

Figure S1 :
Figure S1: Maximum Likelihood phylogenetic tree based on 21 CP amino acid sequences of viruses in the genera Tombusvirus and Dianthovirus.The evolutionary history was inferred by using the Maximum Likelihood method and Le_Gascuel_2008 model [5].The tree with the highest log likelihood (-8026.32) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value.A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 2.1974)).The rate variation model allowed for

Table S1 :
Host range of NRV

Table S2 :
Nucleotide and amino acid changes between the wild type genomic sequence and the full-length cDNA clone.Nucleotide changes leading to amino acid changes are highlighted in bold.

Table S4 :
Percent identity values of pairwise comparisons of RdRp and CP nucleotide (nt) and amino acid (aa) sequences.RdRp: values exceeding 90 % are highlighted in bold, CP: values exceeding 60 % are highlighted in bold